Kieran McCaul

EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer.

Chromosome 11q13 is amplified in about 13% of primary breast cancers. CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding a filamentous actin binding protein, are favoured candidate onocogenes, whereas INT-2 is an unexpressed gene at this locus. In this study we tested the possibility that different regions of this large amplicon could be independently amplified and subsequently defined the phenotype of EMS1 amplified tumours in a series of 961 primary breast carcinomas. Using DNA slot blots, EMS1 was amplified in 15.2% of samples: 5.4% were coamplified for CCND1; 7.9% coamplified for INT-2 and 6.7% showed EMS1 amplification alone. The degree of amplification of CCND1 and INT-2 was highly correlated (P=0.0001). In contrast, no such relationship existed between EMS1 and CCND1 or INT-2 amplification, demonstrating independent amplification of EMS1 in 44% of amplified tumours. EMS1 amplification (⩾twofold increase in copy number) was positively correlated with patient age ⩾50 years (P=0.025), ER positivity (P=0.022), PgR positivity (P=0.018), and was negatively correlated with HER-2/neu (c-erbB2) amplification (P=0.01). In common with CCND1/INT-2, EMS1 amplification was associated with increased risk of relapse in patients with lymph node-negative disease (P=0.028). In contrast, EMS1 and CCND1/INT-2 amplification appeared to confer different phenotypes in ER positive and negative tumours. A ⩾threefold increase in EMS1 copy number was associated with an apparent increased risk of relapse and death in patients with ER negative tumours, but was without effect in ER positive tumours. In contrast, CCND1/INT-2 amplification had no effect in the patients with ER negative tumours but was associated with early relapse in ER positive patients. Thus EMS1 amplification may identify subgroups of breast cancer patients with increased probability of relapse and death distinct from those identified by CCND1/INT-2 amplification. Further studies are required to more clearly determine the functional consequences of EMS1 overexpression and a biological basis for the relationship between EMS1 amplification and phenotype in breast cancer.

Relationship between p53 gene abnormalities and other tumour characteristics in breast‐cancer prognosis

The prognostic significance of p53 gene abnormalities was investigated in 919 primary breast‐cancer patients. p53 expression and tumour‐cell proliferation fraction determined by MIB‐1 count, p53 exon 5 and 6 mutations and HER‐2/neu oncogene amplification were detected by immunohistochemistry, PCR‐SSCP and slot‐blot hybridization, respectively. Increased MIB‐1 count, p53 expression, HER‐2/neu oncogene amplification and p53 mutations were detected in 33%, 29%, 10% and 8% of tumours, respectively. Statistically significant associations were observed between p53 expression or MIB‐1 count and age below 50 years, high‐grade tumours, medullary carcinomas, and absence of hormone receptors. p53 mutations were associated with increased MIB‐1 count, HER‐2/neu oncogene amplification and absence of hormone receptors, but not with age, tumour size or grade, histological subtype, or the number of axillary nodes involved. After a median follow‐up of 66 months, p53 expression was observed to be associated with significant increases in risk of both relapse and death from breast cancer, but not after adjusting for the effect of other parameters. In these analyses, MIB‐1 count, and not HER‐2/neu oncogene amplification, was an independent predictor of prognosis. In node‐negative patients, only p53 exon 5 and 6 mutations and MIB‐1 count were associated with a statistically significant increase in risk of death from breast cancer, independent of tumour size and ER concentration. We conclude that tumour‐cell proliferation fraction, as measured by MIB‐1 count, is the most useful parameter of breast‐cancer prognosis, with the exception of ER, tumour size and the number of axillary nodes involved.

Prospective study of the prognostic significance of epidermal growth factor receptor in primary breast cancer

Bearing in mind the continuing controversy over the prognostic significance of epidermal growth factor receptor (EGF‐r) expression, we investigated its clinical significance prospectively in 345 primary breast cancer patients. The prognostic significance of EGF‐r expression, as measured by a radioligand binding assay, was determined by Coxs multivariate analysis using EGF‐r concentration as a continuous or dichotomous variable. Increased EGF‐r expression was detected in 20–32% of tumours, depending on the cut‐off in concentration used. EGF‐r expression, irrespective of the cut‐off, was not associated with tumour size or grade or the number of axillary nodes involved. There was, however, a strong inverse association between EGF‐r expression and the absence of hormone receptors. After a median follow‐up period of 57 months, multivariate analysis suggested that EGF‐r expression was associated with increases in risk for both relapse and death from breast cancer, even after adjusting for oestrogen receptor (ER) concentration, tumour size and the number of axillary nodes involved. Patients with ER‐positive tumours, which also expressed EGF‐r, had increases in risk for both relapse and death from breast cancer compared with tumours without EGF‐r. Expression of EGF‐r was not a predictor of poor prognosis in either node‐negative or ER‐negative subgroups of patients.

Vimentin expression is not associated with poor prognosis in breast cancer

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immunohistochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB‐1. The vimentin staining pattern was heterogeneous, but in vimentin‐positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high‐grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB‐1 count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increases in risk of relapse or death from breast cancer.

A simple index using video image analysis to predict disease outcome in primary breast cancer

Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour‐suppressor protein and tumour cell proliferation (MIB‐1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow‐up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Coxs univariate analysis indicated that tumour size, number of cancer‐involved nodes, MIB‐1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse‐free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB‐1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut‐point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut‐point of 30% positive nuclear area and MIB‐1 cut‐point of 10% positive nuclear area. Kaplan‐Meier analysis of this image‐based prognostic index identified 4 risk groups with predicted 5‐year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient. Int. J. Cancer (Pred. Oncol.) 84:203–208, 1999.

A SIMPLE INDEX TO PREDICT PROGNOSIS INDEPENDENT OF AXILLARY NODE INFORMATION IN BREAST CANCER

BACKGROUNDnSince the course of breast cancer is often unpredictable, we wished to develop a model using characteristics of the primary tumour alone to predict prognosis.nnnMETHODSnSeveral tumour features were determined, and after a median follow-up duration of 65 months, multivariate analysis identified tumour size and grade, oestrogen receptor concentration, axillary lymph node metastasis and tumour cell proliferation fraction (MIB-1 count) as being independently associated with increases in risk for both relapse and death from breast cancer. A prognostic model was constructed using tumour size and grade, oestrogen receptor concentration and MIB-1 count only. A score of 1 for each was given to tumour size > 20 mm, tumour grade 2 or 3, oestrogen receptor concentration < 10 fmol/mg cytosol protein and MIB-1 count > 9%. Five groups established by assigning a combined score of 0, 1, 2, 3 or 4 for each patient were analysed for their associations with disease-free and overall survivals.nnnRESULTSnThis preliminary model predicted 5-year survival rates of 97, 91, 85, 68 and 50% for the five groups. The model was further simplified by excluding tumour grade from the analysis. The revised model identified four risk groups with predicted 5-year survival rates of 91, 86, 66 and 52%. This model, the Adelaide prognostic index, was also able to identify four risk groups in both node-negative and node-positive patients.nnnCONCLUSIONSnThe Adelaide prognostic index can be used to predict prognosis even in the absence of axillary lymph node information.

TUMOR MICROVASCULARITY HAS NO INDEPENDENT PROGNOSTIC SIGNIFICANCE FOR BREAST CANCER

Summary There is a continuing controversy regarding the value of estimating degree of intra‐tumor vascularity to predict prognosis in breast cancer. In order to resolve this controversy, primary tumors from a cohort of 519 women with breast cancer were analysed to determine whether association exists between degree of vascularity and prognosis. Tumor vascularity was estimated by immunohistochemistry using a monoclonal antibody to the antigen CD31. The tumor area showing the highest degree of vascularity was chosen to score the number of microvessels per unit area. Issues such as the reproducibility of the microvascularity score and its association with tumor parameters including size, histological grade and hormone receptor levels were investigated. Although previously agreed criteria were used, consensus between two pathologists estimations of the degree of vascularity was only moderate. There was no statistically significant association between tumor vascularity score and other currently established parameters of prognosis. After a median follow up of 71 months for axillary node negative patients, there was no association between tumor vascularity score and increased risk of relapse or death from breast cancer. In axillary node positive patients, tumor vascularity score was associated with increased risk of relapse and death from breast cancer. This association was not however independent of other established parameters of prognosis.Abbreviations: DFS, disease‐free survival; IHC, immunohistochemical; OS, overall survival.